Methods for testing for bioaccumulation

ABSTRACT

Methods comprising: providing a test substance, providing two solvents that are substantially immiscible, introducing a known amount of the test substance and known amounts of the two solvents into a single vessel to create a pre-equilibrium sample, adjusting the concentration of the test substance in the pre-equilibrium sample so that the concentration of the test substance is below the critical micelle concentration of both solvents if the concentration of the test substance is not already below the critical micelle concentration, allowing the test substance to equilibrate between the two solvents over time at a substantially constant temperature, determining the equilibrium concentration of the test substance in each of the solvents, and calculating the partition coefficient.

BACKGROUND

The present invention generally relates to testing, measuring, analyzingor predicting bioaccumulation. More particularly, the present inventionrelates to methods for determining the partition coefficient of chemicalsubstances, wherein the partition coefficient may relate to thebioaccumulation of such substances. The methods of the present inventionmay be particularly suitable for measuring or evaluating bioaccumulationof surfactants, although the method may also be used for measuring orevaluating bioaccumulation of other chemical substances.

Bioaccumulation is generally defined as the process through which achemical increases in concentration in a biological organism over timewhen compared to the concentration of the chemical in the environment.Compounds accumulate in living things any time they are taken up andstored faster than they are broken down, metabolized or excreted. Theprocess is normal and can be helpful to life, as in the storage ofvitamins, for example. However, the process can result in injury to lifewhen the equilibrium between exposure and bioaccumulation isoverwhelmed. The extent of bioaccumulation depends on the concentrationof the chemical in the environment, the amount of chemical coming intoan organism from the food, air or water, and the time it takes for theorganism to acquire the chemical and then store, metabolize or degrade,and excrete it. The nature of the chemical itself, such as itssolubility in water and fat, affects its uptake and storage; the abilityof the organism to degrade and excrete the chemical also affects itsuptake and storage. Understanding the dynamic process of bioaccumulationis generally viewed as important in protecting humans and otherorganisms from adverse effects from chemical exposure. Consequently,bioaccumulation has become a critical consideration in the regulation ofchemicals.

Industries using chemicals in the environment are increasingly facedwith regulations concerning bioaccumulation of those chemicals. The oiland gas industry has varying guidelines and regulations in manycountries worldwide relating to chemicals used in the search for andproduction of hydrocarbons from subterranean formations in thosecountries. Some regulations require testing of individual components ofchemicals used. For compliance with such guidelines and regulations, theindustry tests its chemicals and chemical components, often by testmethods or techniques also prescribed, recommended, and/or approved inthe guidelines or regulations.

Guidelines and regulations pertaining to bioaccumulation frequentlyrefer to a value known as a substance's partition coefficient. Thepartition coefficient, often represented as “P” or written in the formof its logarithm to base ten, “log P,” is the ratio of the equilibriumconcentrations of a dissolved substance in a two-phase system consistingof two largely immiscible solvents. For example, the partitioncoefficient of a test substance in solvents n-octanol and water may bewritten as P_(ow), and calculated as the quotient of the equilibriumconcentration of the test substance in n-octanol (C_(n-octanol)) and theequilibrium concentration of the test substance in water (C_(water)),expressed as follows:$P_{ow} = \frac{C_{n\text{-}{octanol}}}{C_{water}}$

The partition coefficient for octanol and water solvents, P_(ow), is akey parameter in studies of the environmental impact of chemicalsubstances. The Organisation for Economic Co-operation and Development's(“OECD”) Guideline for Testing of Chemicals No. 117 states that there isa highly significant relationship between the P_(ow) of substances andtheir bioaccumulation in fish and that P_(ow) is useful in predictingadsorption on soil and sediments and in establishing quantitativestructure-activity relationships for a wide range of biological effects.

One test that has been used to determine the partition coefficient forn-octanol and water is the High Performance Liquid Chromatography (HPLC)Method described in the OECD Guideline for Testing of Chemicals No. 117,incorporated herein in its entirety by reference and available from OECDin Paris, France. This test is performed on analytical columns packedwith a commercially available solid phase containing long hydrocarbonchains (e.g., C₈-C₁₈) chemically bound onto silica. Chemicals injectedonto such a column move along it by partitioning between the mobilesolvent phase and the hydrocarbon stationary phase. The chemicals areretained in proportion to their hydrocarbon-water partition coefficient,with water-soluble chemicals eluted first and oil-soluble chemicalseluted last. This pattern enables the relationship between the retentiontime on a reverse-phase column and the n-octanol/water partitioncoefficient to be established. The partition coefficient is deduced fromthe capacity factor, k, given by the formula:$k = \frac{t_{R} - t_{o}}{t_{o}}$where t_(R) is the retention time of the test substance, and t_(o) isthe dead-time, i.e., the average time an unretained molecule needs topass through the column. Quantitative analytical methods are not neededand only the retention times are measured.

In general, before the P_(ow) value is determined through a test such asthe HPLC Method, a preliminary estimate of P_(ow) is made using knowncalculations. This preliminary estimate may then be used to select whichtest will be used to measure the P_(ow) value more precisely, as certaintests may only be able to reliably determine P_(ow) values within alimited range. For example, the HPLC Method is useful in determiningP_(ow) values when log P_(ow) is in the range between 0 and 7. When thelog P_(ow) value is estimated to be in the range between −2 and 4,another test has been used. That test is the OECD Guideline for Testingof Chemicals No. 107, called the Partition Coefficient(n-octanol/water): Shake-Flask Method, which is incorporated herein inits entirety by reference and available from the OECD in Paris, France.

The Shake-Flask Method is based on the principle that the Nernstpartition law applies at constant temperature, pressure and pH fordilute solutions. OECD Guideline No. 107 states that the law strictlyapplies to a pure substance dispersed between two pure solvents and whenthe concentration of the solute in either phase is not more than 0.01mole per liter. If several different solutes occur in one or both phasesat the same time, the results may be affected. Dissociation orassociation of the dissolved molecules cause deviations from thepartition law.

Neither the HPLC Method nor the Shake-Flask Method is suitable forcalculating the partition coefficients for chemicals that are consideredsurface active, or surfactants. Nevertheless, surfactants are commonlyused in drilling and well treating fluids. It has been previouslydisclosed that a slow-stirring or no-stirring method may be used todetermine the partition coefficients of various surfactants. Accordingto those methods, once a quantity of surfactant has been allowed toequilibrate between two immiscible solvents, the equilibriumconcentration of the surfactant in each solvent may be measured and thepartition coefficient may be calculated. One problem that may ariseduring the performance of these methods is the formation of surfactantmicelles. It is well understood that when molecules (or ions) of a testsubstance such as a surfactant are mixed with a solvent in aconcentration above the critical micelle concentration (“CMC”), themolecules (or ions) may associate to form micelles. The term “micelle”is defined to include any structure that minimizes the contact betweenthe lyophobic (“solvent-repelling”) portion of a test substance moleculeand the solvent, for example, by aggregating the test substancemolecules into structures such as spheres, cylinders, or sheets, whereinthe lyophobic portions are on the interior of the aggregate structureand the lyophilic (“solvent-attracting”) portions are on the exterior ofthe structure. Because the presence of micelles in a solvent may distortthe readings of the equilibrium concentration of the test substance inthe solvent, prior art methods that do not take steps to ensure thatmicelles will not form during equilibration may not yield accuratemeasurements of the equilibrium concentration. Micelles may also beproblematic because, inter alia, micelles may be related to theproblematic emulsification of the substances being tested.

The present invention generally relates to testing, measuring, analyzingor predicting bioaccumulation. More particularly, the present inventionrelates to methods for determining the partition coefficient of chemicalsubstances, wherein the partion coefficient may relate to thebioaccumulation of such substances. The methods of the present inventionmay be particularly suitable for measuring or evaluating bioaccumulationof surfactants, although the method may also be used for measuring orevaluating bioaccumulation of other chemical substances.

In some embodiments, the present invention provides a method comprising:providing a test substance, providing two solvents that aresubstantially immiscible, introducing a known amount of the testsubstance and known amounts of the two solvents into a single vessel tocreate a pre-equilibrium sample, adjusting the concentration of the testsubstance in the pre-equilibrium sample so that the concentration of thetest substance is below the critical micelle concentration of bothsolvents if the concentration of the test substance is not already belowthe critical micelle concentration, allowing the test substance toequilibrate between the two solvents over time at a substantiallyconstant temperature, determining the equilibrium concentration of thetest substance in each of the solvents, and calculating the partitioncoefficient.

The features and advantages of the present invention will be readilyapparent to those skilled in the art. While numerous changes may be madeby those skilled in the art, such changes are within the spirit of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

These drawings illustrate certain aspects of some of the embodiments ofthe present invention, and should not be used to limit or define theinvention.

FIG. 1 is a schematic illustration of a device that may be used todetermine the surface tension of a sample solution.

FIG. 2A is a graphical representation of the surface tension of aexample surfactant A in purified water as a function of surfactantconcentration. The hollow diamonds represent the extrapolated criticalmicelle concentration.

FIG. 2B is a graphical representation of the surface tension of anexample surfactant B in purified water as a function of surfactantconcentration. The hollow diamonds represent the extrapolated criticalmicelle concentration.

FIG. 3A is a graphical representation of the surface tension of anexample surfactant A in purified water pre-saturated with octanol as afunction of surfactant concentration. The hollow diamonds represent theextrapolated critical micelle concentration.

FIG. 3B is a graphical representation of the surface tension of anexample surfactant B in purified water pre-saturated with octanol as afunction of surfactant concentration. The hollow diamonds represent theextrapolated critical micelle concentration.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention generally relates to testing, measuring, analyzingor predicting bioaccumulation. More particularly, the present inventionrelates to methods for determining the partition coefficient of chemicalsubstances, wherein the partition coefficient may relate to thebioaccumulation of such substances. The methods of the present inventionmay be particularly suitable for measuring or evaluating bioaccumulationof surfactants, although the method may also be used for measuring orevaluating bioaccumulation of other chemical substances.

In some embodiments, the present invention provides methods comprisingproviding a test substance; providing two solvents that aresubstantially immiscible; introducing a known amount of the testsubstance and known amounts of the two solvents into a single vessel tocreate a pre-equilibrium sample; adjusting the concentration of the testsubstance in the pre-equilibrium sample so that the concentration of thetest substance is below the critical micelle concentration of bothsolvents, if the concentration of the test substance is not alreadybelow the critical micelle concentration; allowing the test substance toequilibrate between the two solvents over time at a substantiallyconstant temperature; determining the equilibrium concentration of thetest substance in each of the solvents; and calculating the partitioncoefficient.

There may be several potential advantages to the methods of the presentinvention. One such advantage may be these methods calculate partitioncoefficients based on experimental conditions that approximateenvironmental conditions more closely than some prior art methods. Inenvironments in which bioaccumulation is a concern, the concentration ofa surfactant is usually low, e.g., below the critical micelleconcentration (“CMC”) of the surfactant in environmental solvents.Therefore, a method of calculating the partition coefficient of asurfactant based on a pre-equilibrium concentration of the surfactantthat is below the CMC might better simulate bioaccumulation conditionsthan methods in which the initial surfactant concentration exceeds theCMC. Another advantage of the methods of the present invention may bethat by ensuring that the initial concentration of a test substance intwo immiscible solvents is below the CMC of either solvent, it may beexpected that once the test substance is allowed to equilibrate betweenthe solvents, no micelles will be present in either solvent. Thisabsence of micelles may be desirable, because in some cases micelles mayinterfere with the measurement of the equilibrium concentrations.

In some embodiments of the present invention, the two solvents that aresubstantially immiscible may be any two substantially immisciblesolvents that do not adversely interact with each other, with the testsubstance, or with any other substance used in the testing methods.Suitable pairs of solvents may include, but are not limited to, certainoils with certain alcohols, and water with certain alcohols. In someembodiments in which one of the solvents is water, the water may bedistilled or double-distilled. In other embodiments in which one of thesolvents is water, the water may comprise buffered water, salt water,sea water, synthetic sea water, or the like. In some preferredembodiments, the solvents may be water and octanol. In embodiments inwhich octanol is one of the solvents, the octanol is preferably ofanalytical grade or higher.

In some embodiments, one or both of the two substantially immisciblesolvents may be pre-saturated with some quantity of the other solvent.In preferred embodiments, the solvent that is being pre-saturated may bepre-saturated with about 10% of the other solvent for at least 24 hours.That is, for example, a water solvent may be pre-saturated with about10% octanol and/or an octanol solvent may be pre-saturated with about10% water. By way of explanation and not of limitation, pre-saturating asolvent with the other solvent may be advantageous, because no twosolvents are completely immiscible. Pre-saturation may allow a solventto reach equilibrium prior to the equilibration of the test substance.

In general, the test substance for which the partition coefficient iscalculated may be any substance that may be tested for a tendency toaccumulate in a biological organism. In some preferred embodiments, thetest substance may comprise a surface active chemical or surfactant. Aspreviously indicated, in preferred embodiments the step of adjusting theconcentration of the test substance in the pre-equilibrium sample to aconcentration below the CMC of the test substance in both solvents mayprevent the formation of micelles when the test substance is allowed toequilibrate between the two solvents.

In some embodiments, the step of adjusting the concentration of the testsubstance in the pre-equilibrium sample to a concentration below the CMCof the test substance in both solvents may be performed withoutquantifying or otherwise determining the CMC's. For example, individualsolutions that comprise the test substance and one of the solvents inthe same relative amounts as those substances are present in thepre-equilibrium sample may be prepared. Then, a dynamic light scatteringtechnique may be used to detect the presence of micelles in eachindividual solution. Dynamic light scattering techniques are well known,and a person of skill in the art would understand how dynamic lightscattering may be used to detect the presence of micelles. In general,dynamic light scattering involves shining a light through a solution.The presence of micelles in the solution is detected when a point sourcecenter, e.g., a micelle of sufficient size, back-scatters the light. Theamount and pattern of back-scattering may correspond to the numberand/or size of micelles present in the solution. In order to ensure thatthe concentration of the test substance in a solvent is below thecritical micelle concentration, the individual solutions may be seriallydiluted with more of the same solvent until no micelles are detected.The pre-equilibrium sample may then be diluted to the same or lowerdilution.

In some embodiments, the methods of the present invention may comprisethe step of determining the CMC of the test substance in one or both ofthe solvents, e.g., predicting, quantifying, and/or extrapolating theCMC. In preferred embodiments, the step of determining the CMC of thetest substance in either solvent is carried out before the step ofadjusting the concentration of the test substance in the pre-equilibriumsample to a concentration below the CMC of the test substance in thesolvents. In general, the critical micelle concentration of a testsubstance in a solvent may be determined through any method known in theart for determining critical micelle concentration. Suitable methods fordetermining critical micelle concentration include, for example, the useof an inverted vertical pull surface tension method (e.g., a rod-pulltensiometer), and particle size analysis (e.g., photon correlationmeasurements, dynamic light scattering methods, and the like). Inembodiments in which it may be difficult to measure the critical micelleconcentration through surface tension methods, it may be preferable touse particle size analysis techniques, and vice versa. In someembodiments of the present invention in which a solvent comprises a longchain alcohol and the test substance comprises a surfactant, it may beespecially important to experimentally determine the CMC of the testsubstance in the solvent. By way of explanation and not of limitation,this preference arises from the tendency of long chain alcohols tosubstantially lower the CMC of some surfactants, especially ionicmolecules.

In some embodiments in which dynamic light scattering is used todetermine the CMC of a test substance in a solvent, dynamic lightscattering tests are repeated at various concentrations of the testsubstance in the solvent. Then the number of micelles present at thevarious concentrations may be plotted as a function of text substanceconcentration, and the CMC may be extrapolated as the higherconcentration of test substance at which no micelles are detected.Dynamic light scattering may be particularly well suited for determiningthe CMC of a test substance in a solvent when the solvent is highlypurified water. In certain embodiments in which a water solvent has beenpresaturated with octanol, other methods for determining the CMC may bepreferred over dynamic light scattering. By way of explanation and notof limitation, dynamic light scattering may not be the preferred methodof determining the CMC of octanol-saturated water, because the octanolmay distort micelle shape, which can affect the number of micellesdetected. In some embodiments in which an alcohol other than octanol maybe used, that alcohol might also be responsible for distorting micelleshape.

In some embodiments of the present invention, inverted vertical pullsurface tension methods may be used to determine the CMC of a testsubstance in a solvent. In some embodiments, inverted vertical pullsurface tension methods may comprise placing a solution of a testsubstance in a single solvent on a balance; lowering a rod until ittouches the surface of the solution; taring the balance; and then slowlyraising the rod from the solution. The maximum weight reduction thatregisters on the balance as the rod is lifted can be used to calculatethe surface tension of the solution. Methods of calculating the surfacetension from the maximum weight reduction are well known in the art. Forexample, the calculations outlined in Christian et al., LANGMUIR 1998,14, 3126-28 and Christian et al., J. COLLOID AND INTERFACE SCI., 1999,214, 224-30 may be used. In general, these calculations may also takeinto account factors other than the test substance concentration, suchas temperature, density, etc. By repeating the inverted pull surfacetension test at multiple concentrations of the test substance in thesolvent and plotting the calculated surface tensions as a function oftest substance concentration, the CMC may be extrapolated. In general,the CMC is extrapolated as the lowest concentration of test substance atwhich the surface tension would have become constant despite furtherincreases in concentration. One example of a device that may be used tocarry out an inverted pull surface tension method is the rod-pulltensiometer available from Temco, Inc. of Tulsa, Okla. under the tradename EZ TENSIOMETER. FIG. 1 shows a schematic representation of one typeof device that may be suitable for inverted vertical pull surfacetension methods.

In some embodiments, after the two solvents and the test substance areintroduced into a vessel to create a pre-equilibrium sample, the testsubstance is allowed to equilibrate between the two solvents during aslow-stir (or no-sti)r procedure typically conducted in a laboratory orunder laboratory type conditions using laboratory type equipment. Inpreferred embodiments, allowing the test substance to equilibratebetween the two solvents may comprise stirring the pre-equilibriumsample over time. It is believed that in certain embodiments stirringmay reduce the time necessary for equilibration.

In preferred embodiments, the pre-equilibrium sample is stirred at aconstant temperature (e.g., varying by less than 1° C.) and at a slowrate so that emulsions do not begin to form. The temperature selectedmay be any temperature that is below the boiling point of the twosolvents and the test substance. For an octanol-water solvent system, atemperature selected from the range of about 20° C. to about 22° C. ispreferred, although higher temperatures such as about 25° C. mayalternatively be used. Generally, the stirring speed (if any) selectedwill depend on the size and shape of the container and the length of thestirring bar (if a stirring bar is used), as well as the ease with whichthe solvents form emulsions. For surfactants generally, a stir rate thatcreates a vortex no greater than simply reaching from the top to thebottom of the vessel may often be preferred, or more preferably astirring rate that creates a vortex that does not exceed about one-fifththe height of the total fluid column. In some preferred embodiments, theratio of length of the fluid column-to-height of the vortex may be inthe range of about 1 (for the case where the vortex extends from the topto the bottom of the container) to infinity ∞ (for the case of nostirring). In one exemplary embodiment wherein the vessel containing thepre-equilibrium sample has dimensions of about 27.5 mm×70 mm, a vortexof about 15 mm or less may be preferred. This arrangement would providea ratio of length of the fluid column-to-height of the vortex of about4.667. To achieve such a ratio in this example configuration, a stirringrate of about 150 rpm may be used. However, speeds ranging from about 0rpm to about 200 rpm, for example, may be appropriate for use with mostsurfactants in a similarly sized vessel.

After allowing the test substance to begin to equilibrate between thetwo solvents, with or without such stirring, typically for several daysor weeks, and preferably after equilibrium is reached, the concentrationof the test substance in each immiscible layer is measured. For example,the concentration of the test substance in a water layer (C_(water)) andthe concentration of the test substance in an octanol layer(C_(n-octanol)) are measured. From these, a partition coefficient, forexample P_(ow), for the test substance is calculated using the followingformula: $P_{ow} = \frac{C_{n\text{-}{octanol}}}{C_{water}}$

If solvents other than water and n-octanol are used, the heavier solventis substituted for the C_(water) in the formula and the lighter solventis substituted for the C_(n-octanol) in the formula, as follows:$P = \frac{c_{({{lighter}\quad{solvent}})}}{c_{({{heavier}\quad{solvent}})}}$

Samples for this concentration analysis are taken from each solventlayer, for example the water layer and the octanol layer, preferablyimmediately after stirring but in any case before about 1 hour haslapsed after stirring has ceased or after equilibrium is believed tohave been reached. These samples may then be immediately analyzed forcontent and concentration of the test substance or may be stored,preferably at a constant temperature in the range of about 20° C. toabout 22° C. or at room temperature, for later analysis. Measurement ofthe concentration of the test substance may be conducted with anyequipment capable or suitable for this purpose. For example, a lightscattering detector or an ionized mass detector (mass spectroscopy) ispreferred when the test substance is a surfactant as these instrumentsare capable of measuring concentrations of surfactants below theircritical micelle concentration (CMC). When the test substance has nochromaphore for detection, an evaporative light scattering detector ispreferred.

Preferably, such sampling and measurements of the test substanceconcentration in each layer and calculation of the partition coefficientand log P_(ow) value ( or log P value if solvents other than octanol andwater are used) are made periodically during the test to betterascertain when equilibrium is reached. Equilibrium is considered reachedwhen the log P value does not vary more than about 0.3 per measurement,or when the test substance concentration in the layers appears stable.At equilibrium, the P value and the log P value are final values for thetest substance and are available for use in evaluating bioaccumulationof the test substance.

EXAMPLES Example 1

In order to demonstrate how the CMC of a surfactant in highly purifiedwater might be determined through vertical pull surface tension methods,the following experiment was performed. First, two test surfactants,surfactants A and B were selected. Two sample solutions were made bycombining a known quantity of purified water with a known amount ofsurfactant A or B, respectively. The sample solution was placed on thebalance plate of an EZ TENSIOMETER obtained from Temco, Inc. of Tulsa,Okla. A schematic illustration of an example tensiometer configured tomeasure the surface tension of a sample solution is shown in FIG. 1. Inthe test, the metal rod of the tensiometer was lowered until it touchedthe solution and then slowly raised from the solution. Using thereduction in the solution's weight as the rod pulled some of thesolution from the balance, the surface tension of the solution wascalculated. The experiment was repeated with sample solutions comprisingdifferent concentrations of surfactants A and B. The calculated surfacetensions were plotted as a function of concentration, as shown in FIG.2. The extrapolation of the CMCs of surfactants A and B can also be seenin FIG. 2.

Example 2

To investigate the effect of a long chain alcohol on the CMC ofsurfactants A and B, the procedures described in Example 1 were repeatedusing a solvent of purified water that had previously been saturatedwith octanol for 24 hours. The results of these tests are shown in FIG.3, from which it is apparent that the CMC of surfactant A in theoctanol-saturated water was 44% lower than in pure water, and the CMC ofsurfactant B in the octanol-saturated water was 49% lower than in purewater.

Therefore, the present invention is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Theparticular embodiments disclosed above are illustrative only, as thepresent invention may be modified and practiced in different butequivalent manners apparent to those skilled in the art having thebenefit of the teachings herein. Furthermore, no limitations areintended to the details of construction or design herein shown, otherthan as described in the claims below. It is therefore evident that theparticular illustrative embodiments disclosed above may be altered ormodified and all such variations are considered within the scope andspirit of the present invention. In particular, every range of values(of the form, “from about a to about b,” or, equivalently, “fromapproximately a to b,” or equivalently, “from approximately a-b”)disclosed herein is to be understood as referring to the power set (theset of all subsets) of the respective range of values, and set forthevery range encompassed within the broader range of values. Also, theterms in the claims have their plain, ordinary meaning unless otherwiseexplicitly and clearly defined by the patentee.

1. A method comprising: providing a test substance; providing twosolvents that are substantially immiscible; introducing a known amountof the test substance and known amounts of the two solvents into asingle vessel to create a pre-equilibrium sample; adjusting theconcentration of the test substance in the pre-equilibrium sample sothat the concentration of the test substance is below the criticalmicelle concentration of both solvents, if the concentration of the testsubstance is not already below the critical micelle concentration;allowing the test substance to equilibrate between the two solvents overtime at a substantially constant temperature; determining theequilibrium concentration of the test substance in each of the solvents;and calculating the partition coefficient.
 2. The method of claim 1wherein the step of allowing the test substance to equilibrate betweentwo solvents over time at constant temperature comprises stirring thepre-equilibrium sample at a rate that sufficiently slow to avoid thecreation of an emulsion.
 3. The method of claim 1 wherein the testsubstance is a surfactant.
 4. The method of claim 1 wherein the solventsare water and n-octanol.
 5. The method of claim 4 wherein the constanttemperature is in the range of from about 20° C. to about 22° C.
 6. Themethod of claim 1 wherein the constant temperature is below the boilingpoint of the solvents and the test substance.
 7. The method of claim 1wherein the time required for the test substance to equilibrate betweenthe two solvents extends over several days or weeks.
 8. The method ofclaim 4 wherein the step of calculating the partition coefficientcomprises performing the following calculation:$P_{ow} = \frac{C_{n\text{-}{octantol}}}{C_{water}}$ whereinC_(n-octanol) is the equilibrium concentration of test substance in theoctanol solvent, and C_(water) is the equilibrium concentration of thetest substance in the water solvent.